Anti-IgE vaccines

ABSTRACT

The present invention provides compositions and methods for the use of antigenic peptides derived from the Fc portion of the epsilon heavy chain of an IgE molecule as vaccines for the treatment and prevention of IgE-mediated allergic disorders. In particular, the invention provides compositions, methods for the treatment and prevention of IgE-mediated allergic disorders comprising an immunogenic amount of one or more antigenic peptides derived from the CH3 domain or junction of Ch-3/CH4 domain of an IgE molecule and methods for the evaluation of IgE mediated allergies in dogs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 60/228,989, filed Aug. 30, 2000.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of an IgE molecule as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In particular, the present inventionrelates to compositions comprising at least one antigenic peptidederived from the CH3 domain or CH3-CH4 domain junction of an IgEmolecule optionally coupled to a heterologous carrier protein. Thecompositions of the present invention may also comprise an adjuvant. Thecompositions of the present invention induce anti-IgE antibodies, whichbind to soluble (free) IgE in serum and other bodily fluids, but do notcross-link receptor-bound IgE. The present invention further relates tomethods of administering compositions of the invention to animals,preferably mammals and most preferably humans, for the treatment orprevention of IgE-mediated allergic disorders and methods for evaluatingvaccines and other therapies for the treating IgE-mediated allergicdisorders.

BACKGROUND OF THE INVENTION

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (I-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils.

An IgE antibody is a complex molecule consisting of two identical heavychains and two identical light chains held together by disulfide bondsin a “Y” shape-configuration. Each light chain consists of a variable(V_(L)) domain linked to a constant domain (C_(L)), and each heavy chainconsists of a variable domain (Vh) and four constant domains (CH1, CH2,CH3, and CH4, also known as Cε1, Cε2, Cε3, and Cε4; respectively). Thetwo arms of an IgE antibody contain the site at which an IgE antibodybinds to its specific antigen (allergen) and each arm is referred to asa Fab (fragment-antigen-binding) fragment. The tail of an IgE antibodyis termed Fc (fragment-crystalline) as it can form crystals whenseparated from the Fab fragments of the antibody under appropriateexperimental conditions. The Fc fragment of an IgE antibody consists ofthe Cε2, Cε3, and Cε4 domains and contains the biologically activestructures of the IgE antibody (e.g., receptor binding sites).

The production of IgE antibodies requires interactions andcollaborations among three cells; antigen presenting cells (APC), Tlymphocytes (T helper cells; Th) and antibody producing cells (Blymphocytes; B cells). When a foreign substance, an allergen, isintroduced for the first time into the body of subjects (e.g., byinhalation of environmental allergen, ingestion of certain foods, or viathe skin), the allergen is taken up by APCs (e.g., macrophages) whichthen digest or process the allergen into smaller fragments (epitopes).These fragments are displayed on the surface of APCs in association withspecific molecules known as major histocompatibility complex proteins(MhC). The allergen fragment/MhC complex displayed on the surface ofAPCs is recognized and bound by receptors on the surface of specific Tlymphocytes. This recognition and binding event leads to the activationof T lymphocytes and the subsequent expression and secretion ofcytokines such as interleukin-4 (IL-4). These cytokines induce themultiplication, clonal expansion and differentiation of B cells specificfor the allergen in question (i.e., B cell which express on theirsurface immunoglobulin receptors capable of binding to the allergen) andultimately lead to the production of IgE antibodies from these B cells.A portion of the activated T lymphocytes and IgE producing B cellseventually become committed to a pool of cells called T and B memorycells, which are capable of faster recognition of allergen uponsubsequent exposure to the allergen.

In individuals suffering from type I allergic reactions, exposure to anallergen for a second time leads to the production of high levels of IgEantibodies specific for the allergen as a result of the involvement ofmemory B and T cells in the 3-cell interaction required for IgEproduction. The high levels of IgE antibodies produced during the secondexposure lead to cross-linking of IgE receptors on mast cells andbasophils by allergen-bound IgE, which in turn leads to the activationof these cells and the release of the pharmacological mediators that areresponsible for the clinical manifestations of type I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Over the last 15 years, a variety of approaches have been utilized todetermine the FcεRI binding site on IgE. These approaches can beclassified into five different categories. In one approach, smallpeptides corresponding to portions of the Fc part of an IgE moleculewere produced and analyzed for their ability to inhibit IgE from itsreceptors. See, for example, Nakamura et al., EP0263655 published Apr.13, 1988, Burt et al., 1987, European Journal of Immunology, 17:437-440;helm et al., 1988, Nature 331:180-183; helm et al., 1989, PNAS86:9465-9469; Vercelli et al., 1989, Nature 338:649-651; Nio et al,1990, Peptide Chemistry, 2: 203-208; Nio et al., 1993, FEBS Lett.319:225-228; and Nio et al., 1992, FEBS Lett. 314:229-231. Although manyof the peptides described in these studies were shown to inhibit thebinding of IgE to its receptors, different studies reported differentsequences as being responsible for IgE binding.

helm et al. (1988, Nature 331:180-183) identified a 75 amino acidpeptide that spans the junction between C,h2 and C,h3 domains of IgE andshowed that this peptide binds to the IgE receptor with an affinityclose to that of the native IgE molecule. On the other hand, Basu et al.(1993, Journal of Biological Chemistry 268: 13118-13127) expressedvarious fragments from IgE molecules and found that only those fragmentscontaining both the CH3 and CH4 domains were able to bind IgE and thatCH2 domain is not necessary for binding. Vangelista et al. (1999,Journal of Clinical Investigation 103:1571-1578) expressed only the CH3domain of IgE and showed that this domain alone could bind to IgEreceptor and prevent binding of IgE to its receptor. The results of Basuet al. and Vangelista et al. are inconsistent and conflict with those ofhelm et al. cited above.

In a second approach to identify the FcRI binding site on IgE,polyclonal antibodies against peptides corresponding to parts of the CH2domain, CH3 domain or CH4 domain were produced and used to probe forreceptor binding site on IgE (Robertson et al., 1988, Molecular Immunol.25:103-118). Robertson et al. concluded that the amino acid residuesdefined by a peptide derived from the CH4 domain were not likely to beinvolved in receptor binding, whereas amino acid residues defined by apeptide derived from the CH3 domain of IgE were most likely proximal tothe IgE receptor-binding site (amino acids 387-401) however, theanti-CH3 peptide antibodies released histamine from IgE-loaded mastcells indicating that the amino acids defined by the CH3 peptide did notdefine the bona fide IgE receptor-binding site and that anti-CH3 peptideantibodies could cause anaphylaxis.

In a third approach to identify the FcεRI binding site on IgE, severalinvestigators produced IgE mutants in an attempt to identify the aminoacid residues involved in receptor binding (see, e.g., Schwarzbaum etal., 1989, European Journal of Immunology 19:1015-1023; Weetall et al.,1990, Journal of Immunology 145:3849-3854; and Presta et al., 1994,Journal of Biological Chemistry 269:26368-26373). Schwartzbaum et al.demonstrated that a mouse IgE antibody with the point mutation prolineto histidine at amino acid residue 442 in the CH4 domain has a two foldreduced affinity for the IgE receptor. Schwartzbaum et al. concludedthat the CH4 domain of an IgE antibody is involved in IgE binding to itsreceptor, however, Schwartzbaum's conclusion contradict Weetall et al.'sconclusion that the binding of mouse IgE to its high affinity receptorinvolves portions of the CH2 and CH3 domains of the IgE antibody, butnot the CH4 domain. Further, Schwartzbaum et al.'s conclusionscontradict Presta et al.'s conclusion that the amino acid residues ofthe IgE antibody important for binding to the FcεRI are located in theCH3 domain.

In a fourth approach to identify the FcεRI binding site on IgE, chimericIgE molecules were constructed and analyzed for their ability to bind tothe FcεRI. Weetall et al., supra constructed a series of chimeric murineIgE-human IgG molecules and tested their binding to the IgE receptor.Weetall et al., supra concluded that the CH4 domain does not participatein receptor binding and that the CH2 and CH3 domains are both requiredfor binding to the high affinity receptor on mast cells. In anotherstudy, Nissim et al. (1993, Journal of Immunol 150:1365-1374) tested theability of a series of human IgE-murine IgE chimera to bind to the FcεRIand concluded that only the CH3 domain is needed for binding to theFcεRI. The conclusion by Nissim et al. corroborates the conclusion byVangelista et al. that the CH3 domain of IgE alone binds to the FcεRI.However, the conclusions by Nissim et al. and Vangelista et al.contradict the conclusions of Weetall et al. and Robertson et al.

Presta et al., supra produced chimeric human IgG in which the CH2 wasreplaced with CH3 from human IgE. When tested for receptor binding, thischimera bound to the FcεRI albeit with a four-fold reduced affinitycompared with native IgE. The results of Presta et al. appear tocorroborate with the results of Nissim et al., but conflict with thoseof Weetall et al., helm et al., and Basu et. al., cited above. In afurther attempt to define the exact amino acid residues responsible forthe binding of IgE to its receptor, Presta et al. inserted specificamino acid residues corresponding to CH2-CH3 hinge region and threeloops from the CH3 domain of human IgE into their analogous locationswithin human IgG and called these mutants IgGEL. Unfortunately, whenthese IgGEL variants were tested for receptor binding, they exhibitedminimal binding compared to the native IgE or the IgG in which the fulllength CH3 domain replaced the full length CH2 domain.

In a fifth approach to identify the FcεRI binding site on IgE,monoclonal antibodies have been developed and analyzed for their abilityto block IgE binding to the FcεRI. See, for example, Del Prado et al.,1991, Molecular Immunology 28:839-844; Keegan et al., 1991, MolecularImmunology 28:1149-1154; hook et al., 1991, Molecular Immunology28:631-639; Takemoto et al., 1994, Microbiology and Immunology 38:63-71;and Baniyash et al., 1988, Molecular Immunology 25:705-711. Althoughmany monoclonal antibodies have been developed, they have providedlittle information on the bona fide IgE receptor-binding site because inmany cases the amino acid sequence recognized by these monoclonalantibodies have not or could not be identified. Further, the monoclonalantibodies developed may block IgE from binding to its receptor bysteric hindrance or induction of severe conformational changes in theIgE molecule, rather than by the binding and masking of IgE residuesdirectly involved in receptor binding.

It is apparent from the above discussion that approaches that have beendevised to identify the receptor binding site on IgE have producedconflicting results. The difficulty in the identification of the aminoacid residues of IgE responsible for receptor binding could be furthercomplicated by the possibility that the site on IgE used for binding tothe receptor may not be a linear sequence of amino acids, which could bemimicked by a synthetic peptide. Rather, the binding site may be aconformational determinant formed by multiple amino acids that are farapart in the IgE protein sequence which are brought into close proximityonly in the native three-dimensional structure of IgE. Studies with IgEvariants, IgE chimera, and monoclonal anti-IgE antibodies stronglysuggest that the binding site is a conformational determinant.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.high doses of antihistamines and corticosteroids have serious sideeffects such as renal and gastrointestinal toxicities. Thus, othermethods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies (rhuMab E25 andCGP56901) are in advanced stages of clinical development for treatmentof IgE-mediated allergic reactions (see, e.g., Chang, T. W., 2000,Nature Biotechnology 18:157-62). The identity of the amino acid residuesof the IgE molecule recognized by these monoclonal antibodies are notknown and it is presumed that these monoclonal antibodies recognizeconformational determinants on IgE.

Although early results from clinical trials with therapeutic anti-IgEmonoclonal antibodies suggest that these therapies arc effective in thetreatment of atopic allergies, the use of monoclonal antibodies forlong-term treatment of allergies has some significant shortcomings.First, since these monoclonal antibodies were originally produced inmice, they had to be reengineered so as to replace mouse sequences withconsensus human IgG sequences (Presta et al., 1993, The Journal ofImmunology 151:2623-2632). Although this “humanization” process has ledto production of monoclonal antibodies that contain 95% human sequences,there remain some sequences of mouse origin. Since therapy with theseanti-IgE antibodies requires frequent administration of the antibodiesover a long period of time, some treated allergic patients could producean antibody response against the mouse sequences that still remainwithin these therapeutic antibodies. The induction of antibodies againstthe therapeutic anti-IgE would negate the therapeutic impact of theseanti-IgE antibodies at least in some patients. Second, the cost oftreatment with these antibodies will be very high since high doses ofthese monoclonal antibodies are required to induce a therapeutic effect.Moreover, the frequency and administration routes with which theseantibodies have to be administered is inconvenient. A more attractivestrategy for the treatment of IgE-mediated disorders is theadministration of peptides which induce the production of anti-IgEantibodies.

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821)described a strategy involving the use of a peptide derived from the CH4domain of the human IgE coupled to a heterologous carrier protein as anallergy vaccine. However, this peptide has been shown not to induce theproduction of antibodies that react with native soluble IgE. Further,hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccine compositionsbased on fusion of full length CH2-CH3 domains (approximately 220 aminoacid long) to a foreign carrier protein. however, the antibodies inducedby the anti-IgE vaccine compositions proposed by hellman will mostlikely result in anaphylaxis since antibodies against some portions ofthe CH2 and CH3 domains of the IgE molecule have been shown tocross-link the IgE receptor on the surface of mast cell and basophilsand lead to production of mediators of anaphylaxis (see, e.g., Stadleret al., 1993, Int. Arch. Allergy and Immunology 102:121-126). Therefore,a need remains for vaccines for the treatment of IgE-mediated allergicreactions, which do not induce anaphylactic antibodies.

The significant concern over induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (see, e.g.,Rudolf, et al., 1998, Journal of Immunology 160:3315-3321). Kricek etal. (International Publication No. WO 97/31948) screened phage-displayedpeptide libraries with the monoclonal antibody BSWI7 to identify peptidemimotopes that could mimic the conformation of the IgE receptor-bindingsite. These mimotopes could presumably be used to induce polyclonalantibodies that react with free native IgE, but not with receptor-boundIgE as well as block IgE from binding to its receptor. Kricek et al.disclosed peptide mimotopes that are not homologous to any part of theIgE molecule and are thus different from peptides disclosed in thepresent invention.

A major obstacle facing the development of an anti-IgE vaccine is thelack of information regarding the precise amino acids representingnon-anaphylactogenic IgE determinants that could be safely used toimmunize allergic subjects and induce non-anaphylactogenic polyclonalantibodies (i.e., polyclonal anti-IgE antibodies that do not bind toreceptor-bound IgE). The peptide compositions of the present inventionare selected to be non-anaphylactogenic; i.e., the peptide compositionsdo not result in production of anti-IgE antibodies that could causecross-linking of IgE bound to mast cells or basophils. Thus peptides ofthe present invention have superior safety profile and aredifferentiated by sequence composition from disclosed vaccines based onfull-length C2h-CH3 domains.

The safety and efficacy of therapies intended for treatment ofIgE-mediated allergies are usually evaluated in animal models such asmice, rats and dogs. A variety of mouse and rat models have beendeveloped for several types of IgE-mediated allergies such as asthma,atopic dermatitis and food allergies (Xin-Min Li, et al.; J. AllergyClin. Immunol 1999; 103:206-214, Xui-Min et al.; J. Allergy ClinImmunol., 2001, 107:693-702). Although these models have been useful inevaluation of small molecule-based treatment modalities, they are notsuitable for evaluation of vaccine-based treatments. This is because theIgE-derived peptide epitope(s) that are used for development of avaccine for non-rodent species e.g. dogs, can be significantly differentfrom those of mice and rats. Although naturally occurring canine modelsof allergies are available (e.g. Ermel R W, et al.; Laboratory AnimalScience 1997, 47:40-48), these models take a very long time to developand only a limited number of animals are available at one time.Furthermore, once these dogs are used for a vaccine trial, they cannotbe used for further trials. Although dogs can be experimentallysensitized to allergens such as flea allergens (e.g. McDermott, M J, etal.; Molecular Immunology, 2000; 37:361-375), the limitations discussedabove still apply. Thus, an appropriate method to induce high levels ofIgE and clinical signs of Type I hypersensitivity in dogs is needed toallow rapid evaluation of vaccines and other therapies for treatment ofallergies in the desired target species.

Ricin is a lectin found in castor beans which has been found to enhanceIgE production directed against a variety of antigens. For example,administration of ricin in conjunction with an antigen can boost theproduction of IgE in rats that are inherently low in IgE (e.g.Underwood, S L et al.; Immunology. 1995;85:256-61, Underwood, S L etal.; Int Arch Allergy Immunol. 1995;107:119-21 and Diaz-Sanchez D. et.al.; Immunology. 1991;72:297-303). Several studies have determined thatricin enhances IgE responses by preferentially inhibiting a populationof activated CD8+ T lymphocytes. These CD8+ cells are thought to expresscounter regulatory cytokines (e.g. interferon gamma) that down regulatethe Th2 cytokines (IL-4, IL-10, and IL-5) released by CD4+ lymphocytesthat provide class-switching signals for B-lymphocytes to express IgE(Noble A, et al.; Immunology 1993, 80:326 and Diaz-Sanchez, D. et. Al.;Immunology. 1993;78:226-236.). Previous studies also show that IgEresponses to bee venom phospholipase A2 were reduced by 90% in ratsreceiving an adoptive transfer of the immunosuppressive CD8+ Tlymphocytes (Diaz-Sanchez et al.; Immunology 1993, 78:226-236). Comparedto CD4+ cells, this population of regulatory CD8+ T lymphocytes has highaffinity receptors for the ricin lectin. Following entry of the lectininto the activated cell, cellular protein synthesis is inhibitedresulting in killing of the cell. Rats immunized with antigen and ricinshow a dramatic increase in the CD4/CD8 ratio due to a 40% decrease inCD8+ T lymphocytes occurring between 7 and 21 days after immunization((Diaz-Sanchez et al.; Immunology 1993, 78:226-236).

Thus to facilitate and accelerate the development of allergy models,there is a need as provided in the method of the present invention, forinduction of high levels of IgE and concomitant induction of clinicalsigns of allergies in normal dogs following simultaneous exposure toallergens and ricin. This method utilizes normal dogs, which are readilyavailable, and results in sensitization of the majority of dogs in arelatively short period of time.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of an IgE molecule as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In particular, the invention providescompositions for the treatment and prevention of IgE-mediated allergicdisorders comprising an immunogenic amount of one or more antigenicpeptides derived from the CH3 domain of an IgE molecule.

Preferably, compositions of the present invention comprise animmunogenic amount of one or more antigenic peptides comprising theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. FIGS. 3-9 depict suchdog CH3/CH4 peptide sequences. Further preferred compositions of thepresent invention comprise an immunogenic amount of one or moreantigenic peptides comprising the amino acid sequence of SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14. FIGS. 10-16 depict such human CH3/CH4 peptidesequences.

The antigenic peptides can be supplied by direct administration orindirectly as “pro-drug” using somatic cell gene therapy.

The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3 domain of an IgE molecule and one or morepharmaceutically acceptable carriers. In one embodiment, apharmaceutical composition of the invention comprises an immunogenicallyeffective amount of one or more antigenic peptides derived from the CH3domain of an IgE molecule and one or more pharmaceutically acceptablecarriers. In another embodiment, a pharmaceutical composition of theinvention comprises an immunogenically effective amount of one or moreantigenic peptides derived from the junction of the CH3 and CH4 domainsof an IgE molecule and one or more pharmaceutically acceptable carriers.

In a particular embodiment, a pharmaceutical composition of theinvention comprises one or more pharmaceutical carriers and animmunogenically effective amount of one or more antigenic fusionproteins comprising an antigenic peptide derived from the CH3 domain ofan IgE molecule and a heterologous carrier protein. In anotherparticular embodiment, a pharmaceutical composition of the inventioncomprises one or more pharmaceutical carriers and an immunogenicallyeffective amount of one or more antigenic fusion proteins comprising anantigenic peptide derived from the junction of the CH3 and CH4 domainsof an IgE molecule and a heterologous carrier protein.

In a preferred embodiment, a pharmaceutical composition of the inventioncomprises one or more pharmaceutical carriers and an immunogenicallyeffective amount of one or more antigenic peptides comprising the aminoacid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In another preferredembodiment, a pharmaceutical composition of the present inventioncomprises one or more pharmaceutical carriers and an antigenic fusionprotein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7 coupled to a heterologous carrier protein.

In a further preferred embodiment, a pharmaceutical composition of theinvention comprises one or more pharmaceutical carriers and animmunogenically effective amount of one or more antigenic peptidescomprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.In another preferred embodiment, a pharmaceutical composition of thepresent invention comprises one or more pharmaceutical carriers and anantigenic fusion protein comprising the amino acid sequence of SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, or SEQ ID NO: 14 coupled to a heterologous carrier protein.

The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3 domain of an IgE molecule, apharmaceutically acceptable carrier, and an adjuvant. Adjuvantsencompass any compound capable of enhancing an immune response to anantigen. Examples of adjuvants which may be effective, include, but arenot limited to: aluminum hydroxide, monophosphoryl lipid A(MPLA)-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1″-2″-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,simple immunostimulatory oligonucleotides, cytokines such as IL-12, IL-2or IL-1, saponins, and microbial toxins such as cholera toxin, heatlabile toxin and genetically altered derivatives of them.

In one embodiment, a pharmaceutical composition of the inventioncomprises an immunogenically effective amount of one or more antigenicpeptides derived from the CH3 domain of an IgE molecule, apharmaceutically acceptable carrier, and an adjuvant. In anotherembodiment, a pharmaceutical composition of the invention comprises animmunogenically effective amount of one or more antigenic peptidesderived from the junction of the CH3 and CH4 domains of an IgE molecule,a pharmaceutically acceptable carrier, and an adjuvant. In anotherembodiment, a pharmaceutical composition of the invention comprises apharmaceutical carrier, an adjuvant and an immunogenically effectiveamount of one or more antigenic fusion proteins comprising an antigenicpeptide derived from the CH3 domain of an IgE molecule and aheterologous carrier protein. In yet another embodiment, apharmaceutical composition of the invention comprises a pharmaceuticalcarrier, an adjuvant and an immunogenically effective amount of one ormore antigenic fusion proteins comprising an antigenic peptide derivedfrom the junction of the CH3 and CH4 domains of an IgE molecule and aheterologous carrier protein.

In a preferred embodiment, a pharmaceutical composition of the inventioncomprises a pharmaceutical carrier, an adjuvant and an immunogenicallyeffective amount of one or more antigenic peptides comprising of theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In another preferredembodiment, a pharmaceutical composition of the present inventioncomprises a pharmaceutical carrier, an adjuvant, and an immunogenicallyeffective amount of one or more antigenic fusion proteins comprising theamino acid sequence of SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 coupled to aheterologous carrier protein.

In a further preferred embodiment, a pharmaceutical composition of theinvention comprises a pharmaceutical carrier, an adjuvant and animmunogenically effective amount of one or more antigenic peptidescomprising of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:14. In yet another preferred embodiment, a pharmaceutical composition ofthe present invention comprises a pharmaceutical carrier, an adjuvant,and an immunogenically effective amount of one or more antigenic fusionproteins comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ IDNO: 14 coupled to a heterologous carrier protein.

The present invention also provides methods of administeringcompositions of the invention to animals, preferably mammals and mostpreferably humans for the treatment or prevention of IgE-mediatedallergic disorders. The compositions of the present invention are insuitable formulation to be administered to animals, preferably mammalssuch as companion animals (e.g., dogs, cats, and horses) and livestock(e.g., cows and pigs), and most preferably humans. The compositions ofthe invention are administered in an amount effective to elicit animmune response, for example, the production of polygonal antibodieswith specificity for an IgE molecule. In one embodiment, thecompositions of the invention are administered in an amount effective toinduce the production of polyclonal antibodies with specificity for theFc portion of an IgE molecule required for IgE to bind to its receptor(i.e., the CH3 domain of an IgE molecule). In a preferred embodiment,the compositions of present invention are administered in an amounteffective to induce the production of anti-IgE antibodies which bind tosoluble (free) IgE in serum and other bodily fluids, prevent IgE frombinding to its high affinity receptors on mast cells and basophils, anddo not cross-link receptor-bound IgE. Accordingly, the compositions ofthe invention are administered in an amount effective to induce theproduction of polyclonal antibodies which do not induce anaphylaxis forthe treatment or prevention of IgE-mediated allergic disorders.

The present invention also provides a method for evaluating the effectof anti-IgE vaccines in dogs which comprises sensitization of the dogsto an allergen by concurrent administration of the allergen and ricin inamountsl sufficient to induce hypersensitivity in the dogs, followed bychallenge with the allergen and observation of the resulting sensitivityof the dogs to the challenge allergen. Specific embodiments of themethod include those wherein the allergen is a flea allergen or a foodallergen such as an ascaris allergen. In one embodiment of the methodthe hypersentivity is type I hpersensitivity. In another embodiment ofthe method sensitization results in higher levels of IgE in thehypersensitized dogs than found in non-hypersensitized dogs.

The present invention further provides a method for inducing high levelsof IgE and clinical signs of hypersensitivity in dogs for evaluating theeffect of anti-IgE vaccines in the dogs which comprises: sensitizationof the dogs to an allergen sufficient to induce hypersensitivity in thedogs by concurrent administration of amounts of the allergen and ricinsufficient to to the dogs, followed by challenge with the allergen andobservation of the resulting sensitivity of the dogs to the challengeallergen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the ELISA reactivity of sera obtained from rabbitsimmunized with RBS peptides 1-4 (SEQ ID 1-4; respectively) against therespective RBS peptides coated onto neutravidin plates.

FIG. 2 depicts the ELISA reactivity of sera obtained from rabbitsimmunized with RBS peptides 1-4 (SEQ ID 1-4, respectively) against thefull-length canine IgE protein.

FIGS. 3-9 depict dog CH3/CH4 peptide sequences.

FIG. 3 depicts SEQ ID NO: 1; Dog CH3/CH4 peptide sequence.

FIG. 4 depicts SEQ ID NO: 2; Dog CH3/CH4 peptide sequence.

FIG. 5 depicts SEQ ID NO: 3; Dog CH3/CH4 peptide sequence.

FIG. 6 depicts SEQ ID NO: 4; Dog CH3/CH4 peptide sequence.

FIG. 7 depicts SEQ ID NO: 5; Dog CH3/CH4 peptide sequence.

FIG. 8 depicts SEQ ID NO: 6; Dog CH3/CH4 peptide sequence.

FIG. 9 depicts SEQ ID NO: 7; Dog CH3/CH4 peptide sequence.

FIGS. 10-16 depict human CH3/CH4 peptide sequences.

FIG. 10 depicts SEQ ID NO: 8; human CH3/CH4 peptide sequence.

FIG. 11 depicts SEQ ID NO: 9; human CH3/CH4 peptide sequence.

FIG. 12 depicts SEQ ID NO: 10; human CH3/CH4 peptide sequence.

FIG. 13 depicts SEQ ID NO: 11; human CH3/CH4 peptide sequence.

FIG. 14 depicts SEQ ID NO: 12; human CH3/CH4 peptide sequence.

FIG. 15 depicts SEQ ID NO: 13; human CH3/CH4 peptide sequence.

FIG. 16 depicts SEQ ID NO: 14; human CH3/CH4 peptide sequence.

FIG. 17 depicts SEQ ID NO: 15; Dog CH3/CH4 nucleotide sequence.

FIG. 18 depicts SEQ ID NO: 16; Dog CH3/CH4 nucleotide sequence.

FIG. 19 depicts SEQ ID NO: 17; Dog CH3/CH4 nucleotide sequence.

FIG. 20 depicts SEQ ID NO: 18; Dog CH3/CH4 nucleotide sequence.

FIG. 21 depicts SEQ ID NO: 19; Dog CH3/CH4 nucleotide sequence.

FIG. 22 depicts SEQ ID NO: 20; Dog CH3/CH4 nucleotide sequence.

FIG. 23 depicts SEQ ID NO: 21; Dog CH3/CH4 nucleotide sequence.

FIG. 24 depicts SEQ ID NO: 22; Dog CH3/CH4 nucleotide sequence.

FIG. 25 depicts SEQ ID NO: 23; Dog CH3/CH4 nucleotide sequence.

FIG. 26 depicts SEQ ID NO: 24; Dog CH3/CH4 nucleotide sequence.

FIG. 27 depicts SEQ ID NO: 25; Dog CH3/CH4 nucleotide sequence.

FIG. 28 depicts SEQ ID NO: 26; Dog CH3/CH4 nucleotide sequence.

FIG. 29 depicts SEQ ID NO: 27; Dog CH3/CH4 nucleotide sequence.

FIG. 30 depicts SEQ ID NO: 28; Dog CH3/CH4 nucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of an IgE molecule as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In particular, the present inventionprovides compositions comprising an immunogenic amount of an antigenicpeptide derived from the CH3 domain of an IgE molecule effective fortreatment or prevention of an IgE-mediated allergic disorder.Preferably, compositions of the present invention comprise animmunogenic amount of one or more antigenic peptides comprising theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 (FIGS. 3-9).Further preferred compositions of the present invention comprise animmunogenic amount of one or more antigenic peptides comprising theamino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 0, or SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:14 (FIGS. 10-16).

The antigenic peptides of the present invention comprise an amino acidsequence of the CH3 domain of an IgE molecule or a fragment thereof andinduce the production of anti-IgE antibodies, which are notanaphylactic. The present invention also encompasses antigenic peptidescomprising an amino acid sequence of the junction of the CH3 and CH4domains of an IgE molecule, which induce anti-IgE antibodies that arenot anaphylactic. In particular, the antigenic peptides of the presentinvention induce the production of anti-IgE antibodies which bind tosoluble (free) IgE in serum and other bodily fluids, prevent IgE frombinding to its high affinity receptors on mast cells and basophils, anddo not cross-link receptor-bound IgE. The antigenic peptides of thepresent invention may be coupled to one or more heterologous peptides.The antigenic peptides of the invention can be supplied by directadministration or indirectly as “pro-drugs” using somatic cell genetherapy.

In one embodiment, an antigenic peptide of the invention comprises theentire CH3 domain of an IgE molecule of any species. In anotherembodiment, an antigenic peptide of the invention comprises a fragmentof the CH3 domain of an IgE molecule of any species, wherein thefragment is at least five amino acid residues long, preferably at least10 amino acid residues long, more preferably at least 15 amino acidresidues long, at least 20 amino acid residues long, at least 25 aminoacid residue long, or at least 30 amino acid residues long. In apreferred embodiment, an antigenic peptide of the invention comprises anamino acid sequence of a fragment of the CH3 domain of an IgE moleculethat is between 28 and 31 amino acid residues. In another preferredembodiment, an antigenic peptide of the present invention comprises anamino acid sequence of a fragment of the CH3 domain of an IgE moleculethat does not possess two cysteine amino acid residues separated by 21amino acid residues, 22 amino acid residues, 23 amino acid residues, 24amino acid residues, or 25 amino acid residues. In a specificembodiment, an antigenic peptide of the invention comprises the junctionof the CH3 and CH4 domains of an IgE molecule or a fragment thereof,wherein the fragment is at least five amino acid residues long,preferably at least 10 amino acid residues long, more preferably atleast 15 amino acid residues long, at least 20 amino acid residues long,at least 25 amino acid residue long, or at least 30 amino acid residueslong.

In a preferred embodiment, an antigenic peptide of the present inventioncomprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 (FIGS.3-9). In another preferred embodiment, an antigenic peptide of theinvention comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ IDNO: 14 (FIGS. 10-16).

The present invention also provides antigenic fusion proteins comprisingan antigenic peptide and a heterologous carrier protein. In a specificembodiment, an antigenic fusion protein comprises the entire CH3 domainof an IgE molecule and a heterologous carrier protein. In anotherspecific embodiment, an antigenic fusion protein comprises a fragment ofan IgE molecule coupled to a heterologous carrier protein, wherein thefragment of the CH3 domain is at least five amino acids long, preferablyat least 10 amino acid residues long, more preferably at least 15 aminoacid residues long, at least 20 amino acid residues long, at least 25amino acid residue long, or at least 30 amino acid residues long. Inanother embodiment, an antigenic fusion protein of the present inventioncomprises the junction of the CH3 and CH4 domains of an IgE molecule ora fragment thereof coupled to a heterologous carrier protein. In apreferred embodiment, an antigenic fusion protein of the presentinvention comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7. In another preferred embodiment, an antigenic fusion protein of thepresent invention comprises the amino acid sequence of SEQ ID NO: 8, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, orSEQ ID NO: 14.

The present invention also provides antigenic peptides or antigenicfusion proteins comprising an amino acid sequence derived from a CH3domain of an IgE molecule in which one or more amino acid substitutions,additions or deletions has been introduced. Mutations can be introducedby standard techniques known to those of skill in the art.

For example, one or more mutations at the nucleotide level which resultin one or more amino acid mutations can be introduced by site-directedmutagenesis or PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for their ability to induceanti-IgE antibodies which do not cause anaphylaxis.

The present invention also provides methods for treating or preventingIgE-mediated allergic disorders in animals, preferably mammals and mostpreferably humans, comprising administering pharmaceutical compositions,which do not induce anaphylaxis. The pharmaceutical compositions to beadministered in accordance with the methods of the present inventionencompass antigenic peptides derived from the CH3 domain of IgEmolecule. The pharmaceutical compositions to be administered inaccordance with the methods of the present invention also include: (i)recombinant antigenic peptides comprising an amino acid sequence of aCH3 domain of an IgE molecule or a fragment thereof; (ii) recombinantantigenic fusion proteins comprising an amino acid sequence of a CH3domain of an IgE molecule or a fragment thereof and a heterologouscarrier protein; (iii) recombinant antigenic peptides comprising anamino acid sequence of a junction of the CH3 and CH4 domains of an IgEmolecule or a fragment thereof; (iv) recombinant antigenic fusionproteins comprising an amino acid sequence of a junction of the CH3 andCH4 domains of an IgE molecule or a fragment thereof; (v) plasmidcompositions comprising polynucleotides encoding an antigenic peptidehaving an amino acid sequence of a CH3 domain of an IgE molecule or afragment thereof; (vi) plasmid compositions comprising polynucleotidesencoding for antigenic fusion proteins comprising an amino acid sequenceof a CH3 domain of an IgE molecule or a fragment thereof and aheterologous carrier protein; (vii) plasmid compositions comprisingpolynucleotides encoding an antigenic peptide having an amino acidsequence of a junction of the CH3 and CH4 domains of an IgE molecule ora fragment thereof; and (viii) plasmid compositions comprisingpolynucleotides encoding for antigenic fusion proteins comprising anamino acid sequence of a junction of the CH3 and CH4 domains of an IgEmolecule or a fragment thereof.

In one embodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic peptides comprising the amino acidsequence of the entire CH3 domain of an IgE molecule. In anotherembodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic peptides comprising the amino acidsequence of a fragment of the CH3 domain of an IgE molecule, wherein thefragment is at least five amino acid residues long, preferably at least10 amino acid residues long, more preferably at least 15 amino acidresidues long, at least 20 amino acid residues long, at least 25 aminoacid residue long, or at least 30 amino acid residues long. In apreferred embodiment, a pharmaceutical composition of the presentinvention comprises one or more antigenic peptides comprising the aminoacid sequence of a fragment of the CH3 domain of an IgE molecule that isbetween 28 and 31 amino acid residues. In another preferred embodiment,pharmaceutical compositions of the present invention comprise one ormore antigenic peptides comprising the amino acid sequence of a fragmentof the CH3 domain of an IgE molecule that does not possess two cysteineamino acid residues separated by 21 amino acid residues, 22 amino acidresidues, 23 amino acid residues, 24 amino acid residues, or 25 aminoacid residues. In accordance with these embodiments, the pharmaceuticalcompositions may further comprise an adjuvant.

In a specific embodiment, a pharmaceutical composition of the presentinvention comprises one or more antigenic peptides comprising the aminoacid sequence of a junction of the CH3 and CH4 domains of an IgEmolecule or a fragment thereof. In accordance with this embodiment, thepharmaceutical composition may further comprise an adjuvant. Preferably,the antigenic peptide comprising the amino acid sequence of a junctionof the CH3 and CH4 domains of an IgE molecule or a fragment thereof isbetween 28 and 31 amino acid residues.

The present invention also provides pharmaceutical compositionscomprising one or more antigenic fusion proteins. In a specificembodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic fusion proteins comprising an antigenicpeptide of the invention and a heterologous carrier protein. Inaccordance with this embodiment, the pharmaceutical composition mayfurther comprise an adjuvant.

As used herein the term “heterologous carrier protein” refers to aprotein which does not possess high homology to a protein found in thespecies that is receiving a composition of the invention and elicits animmune response. A protein possesses high homology if it is at least 75%identical, more preferably at least 85% identical or at least 90%identical to a protein as determined by any known mathematical algorithmutilized for the comparison of two amino acid sequences (see, e.g.,Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268;Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877;Torellis and Robotti, 1994, Comput. Appl. Biosci. 10: 3-5; and Pearsonand Lipman, 1988, Proc. Natl. Acad. Sci. 85: 2444-8). Preferably, thepercent identity of two amino acid sequences is determined by BLASTprotein searches with the XBLAST program, score=50, wordlength=3.Examples of heterologous carrier proteins include, but are not limitedto, KLh, PhoE, rmLT, TraT, or gD from BhV-1 virus.

A heterologous carrier protein can be fused to the N-terminus orC-terminus of an antigenic peptide of the invention. Antigenic fusionproteins of the invention can be produced by techniques known to thoseof skill in the art, for example, by standard recombinant DNAtechniques. For example, a nucleotide sequence encoding an antigenicfusion protein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification ofnucleotide fragments can be carried out using anchor primers which giverise to complementary overhangs between two consecutive nucleotidefragments which can subsequently be annealed and reamplified to generatea nucleotide sequence encoding an antigenic fusion protein (see, e.g.,Ausubel et al., infra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding an antigenic peptide of theinvention can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the antigenic peptide of theinvention. Further, a heterologous carrier protein can be fused to anantigenic peptide by chemical methods known to those of skill in theart.

In a specific embodiment, a pharmaceutical composition of the presentinvention provides an antigenic peptide having an amino acid sequencecomprising amino acid residues of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In anotherembodiment, a pharmaceutical composition of the present inventionprovides an antigenic fusion protein comprising the amino acid sequenceof SEQ ID NO: 1 coupled to a heterologous carrier protein, the aminoacid sequence of SEQ ID NO: 2 coupled to a heterologous carrier protein,the amino acid sequence of SEQ ID NO: 3 coupled to a heterologouscarrier protein, the amino acid sequence of SEQ ID NO: 4 coupled to aheterologous carrier protein, the amino acid sequence of SEQ ID NO: 5coupled to a heterologous carrier protein, the amino acid sequence ofSEQ ID NO: 6 coupled to a heterologous carrier protein, or the aminoacid sequence of SEQ ID NO: 7 coupled to a heterologous carrier protein.In another specific embodiment, a pharmaceutical composition of thepresent invention provides an antigenic fusion protein having the aminoacid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In anotherembodiment, a pharmaceutical composition of the present inventionprovides an antigenic fusion protein comprising the amino acid sequenceof SEQ ID NO: 8 coupled to a heterologous carrier protein, the aminoacid sequence of SEQ ID NO: 9 coupled to a heterologous carrier protein,the amino acid sequence of SEQ ID NO: 10 coupled to a heterologouscarrier protein, the amino acid sequence of SEQ ID NO: 11 coupled to aheterologous carrier protein, the amino acid sequence of SEQ ID NO: 12coupled to a heterologous carrier protein, the amino acid sequence ofSEQ ID NO: 13 coupled to a heterologous carrier protein, or the aminoacid sequence of SEQ ID NO: 14 coupled to a heterologous carrierprotein. In accordance with these embodiments, the pharmaceuticalcompositions may further comprise an adjuvant.

The pharmaceutical compositions of the present invention are in suitableformulation to be administered to animals such as companion animals(e.g., dogs and cats) and livestock (e.g., pigs, cows and horses) andhumans for the treatment or prevention of IgE-mediated allergicdisorders. Preferably, a pharmaceutical composition of the inventioncomprises an antigenic peptide derived from the CH3 domain of the IgEmolecule of the same species receiving the antigenic peptide to treat orprevent an IgE-mediated allergic disorder. IgE-mediated allergicdisorders include, but are not limited to, asthma, allergic rhinitis,gastrointestinal allergies such as food allergies, eosinophilia,conjunctivitis, glomerular nephritis and graft-versus-host disease. Thepharmaceutical compositions of the invention are administered to asubject (an animal) in an amount effective for the treatment, preventionor inhibition of IgE-mediated allergic disorders, or an amount effectivefor inducing an anti-IgE immune response (i e., the production ofanti-IgE polyclonal antibodies) that is not anaphylactic, or an amounteffective for inhibiting or reducing the release of vasoactivesubstances such as histamine, or an amount effective for alleviating oneor more symptoms associated with an IgE-mediated allergic disorder.

The pharmaceutical compositions of the invention can be used with anyknown method of treating IgE-mediated allergic disorders. In oneembodiment, one or more pharmaceutical compositions of the invention andone or more antihistamines are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In anotherembodiment, one or more pharmaceutical compositions of the invention andone or more corticosteroids are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In yetanother embodiment, one or more pharmaceutical compositions of theinvention and one or more anti-IgE monoclonal antibodies (e.g., BSW17)are administered to an animal for the treatment or prevention of anIgE-mediated allergic disorder.

The present invention also comprises polynucleotide sequences encodingthe antigenic peptides or antigenic fusion proteins of the invention.The present invention comprises nucleic acid molecules comprisingdifferent polynucleotide sequences due to the degeneracy of the geneticcode which encode identical antigenic peptides and antigenic fusionproteins. The present invention encompasses antigenic peptidescomprising an amino acid sequence of a CH3 domain of an IgE molecule ora fragment thereof encoded by the polynucleotide sequence of anyspecies. The polynucleotide sequence of a CH3 domain of an IgE moleculecan be obtained from scientific literature, Genbank, or using cloningtechniques known to those of skill in the art. In particular, thepresent invention encompasses polynucleotide sequences encoding humanand canine the CH3 domain of an IgE molecule the disclosed in GenbankAccession Number AAB59424.1 and AAA56797.1; respectively, areincorporated herein by reference. The present invention furtherencompasses antigenic peptides comprising an amino acid sequence of ajunction of the CH3 and CH4 domains of an IgE molecule or a fragmentthereof encoded by the polynucleotide sequence of any species. Thepolynucleotide sequence of a junction of the CH3 and CH4 domains of anIgE molecule can be obtained from scientific literature, Genbank, orusing cloning techniques known to those of skill in the art.

The present invention also encompasses antigenic fusion proteinscomprising an antigenic peptide encoded by a polynucleotide sequence ofany species and a heterologous carrier protein encoded by apolynucleotide sequence of a different species from the antigenicpeptide. The polynucleotide sequence of a heterologous carrier proteincan be obtained from scientific literature, Genbank, or using cloningtechniques known to those of skill in the art.

The polynucleotide sequence encoding an antigenic peptide or anantigenic fusion protein of the invention can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. The necessary transcriptional and translationalsignals can also be supplied by the native IgE genes or its flankingregions. A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining polynucleotides encoding antigenic peptides or antigenicfusion proteins, and appropriate transcriptional and translationalcontrol signals. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of the nucleic acid sequence encoding an antigenic peptide oran antigenic fusion protein of the invention may be regulated by asecond nucleic acid sequence so that the antigenic peptide or theantigenic fusion protein is expressed in a host transformed with therecombinant DNA molecule. For example, expression of an antigenicpeptide or an antigenic fusion protein of the invention may becontrolled by any promoter or enhancer element known in the art.Promoters which may be used to control the expression of an antigenicpeptide or an antigenic fusion protein of the invention include, but arenot limited to, the Cytomeglovirus (CMV) immediate early promoterregion, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290: 304-310), the promoter contained in the 3″ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296: 39-42);prokaryotic expression vectors such as the 3-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731),or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also ““Useful proteins from recombinant bacteria”” inScientific American, 1980, 242: 74-94; plant expression vectorscomprising the nopaline synthetase promoter region (herrera-Estrella etal., Nature 303: 209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(herrera-Estrella et al., 1984, Nature 310: 115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz etal., 1986, Cold Spring harbor Symp. Quant. Biol. 50: 399-409; MacDonald,1987, hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (hanahan, 1985, Nature 315: 115-122);immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature318: 533-538; and Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45: 485-495); albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1: 268-276); alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5: 1639-1648; and hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in theliver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315: 338-340; and Kollias et al., 1986, Cell 46: 89-94);myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active inskeletal muscle (Sani, 1985, Nature 314: 283-286); swine alpha-skeletalactin control region which is active in muscle (Reecy, M. et al., 1998,Animal Biotechnology 9: 101-120);and gonadotropic releasing hormone genecontrol region which is active in the hypothalamus (Mason et al., 1986,Science 234: 1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to an antigenic peptide-encoding nucleic acid, one ormore origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene). In another specificembodiment, a vector is used that comprises a promoter operably linkedto an antigenic fusion protein-encoding nucleic acid, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

Expression vectors containing gene inserts can be identified by threegeneral approaches: (a) nucleic acid hybridization; (b) presence orabsence of “marker” gene functions; and (c) expression of insertedsequences. In the first approach, the presence of antigenicpeptide-encoding polynucleotides or antigenic fusion protein-encodingpolynucleotides inserted in an expression vector(s) can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to the inserted polynucleotide sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of the gene(s) in the vector(s). For example, ifa nucleic acid molecule encoding an antigenic peptide or an antigenicfusion protein is inserted within the marker gene sequence of thevector, recombinants containing the nucleic acid molecule encoding theantigenic peptide or the antigenic fusion protein insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe gene product expressed by the recombinant. Such assays can be based,for example, on the physical or functional properties of an antigenicpeptide or an antigenic fusion protein in in vitro assay systems, e.g.,binding of an antigenic peptide or an antigenic fusion protein with ananti-IgE antibody.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

The term “host cell” as used herein refers not only to the particularsubject cell into which a recombinant DNA molecule is introduced butalso to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers; thus, expression of thegenetically engineered may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce an unglycosylated core protein product.Expression in yeast will produce a glycosylated product. Expression inmammalian cells can be used to ensure “native” glycosylation of anantigenic peptide or antigenic fusion protein of the invention.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantigenic peptide or an antigenic fusion protein of the invention may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express an antigenic peptide or an antigenicprotein of the invention. Such engineered cell lines may be particularlyuseful in the screening and evaluation of anti-IgE antibodies or otheragents (e.g., organic molecules, inorganic molecules, organic/inorganiccomplexes, polypeptides, peptides, peptide mimics, polysaccharides,saccharides, glycoproteins, nucleic acids, DNA and RNA strands andoligonucleotides, etc.) that affect binding of an IgE molecule to itsreceptor.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Proc. Natl. Acad. Sci. USA 77: 3567; O'hare et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistanceto hygromycin (Santerre et al., 1984, Gene 30: 147) genes.

In a specific embodiment, one or more nucleic acid molecules comprisinga polynucleotide sequence encoding an antigenic peptide of theinvention, are administered to treat or prevent IgE-mediated allergicdisorders, by way of gene therapy. In another specific embodiment, oneor more nucleic acid molecules comprising a polynucleotide sequenceencoding an antigenic fusion protein, are administered to treat orprevent IgE-mediated allergic disorders, by way of gene therapy. In yetanother specific embodiment, one or more nucleic acid moleculescomprising a polynucleotide sequence encoding an antigenic peptide ofthe invention, and one or more nucleic acid molecules comprising apolynucleotide sequence encoding an antigenic fusion protein of theinvention are administered to treat or prevent IgE-mediated allergicdisorders, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antigenic peptides or antigenicfusion proteins that mediate a therapeutic effect by eliciting an immuneresponse such as the production of anti-IgE antibodies.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596;Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62: 191-217; May, 1993, TIBTECh 11(5):155-215).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler,1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,NY.

In a preferred aspect, a pharmaceutical composition comprises nucleicacid sequences encoding an antigenic peptide of the invention, saidnucleic acid sequences being part of expression vectors that express theantigenic peptide in a suitable host. In particular, such nucleic acidsequences have promoters operably linked to the antigenic peptide codingregions, said promoters being inducible or constitutive, and,optionally, tissue-specific. In another preferred aspect, apharmaceutical composition comprises nucleic acid sequences encoding anantigenic fusion protein of the invention, said nucleic acid sequencesbeing part of expression vectors that express the antigenic fusionprotein in a suitable host. In particular, such nucleic acid sequenceshave promoters operably linked to the antigenic fusion protein codingregions, said promoters being inducible or constitutive, and,optionally, tissue-specific. In another particular embodiment, nucleicacid molecules are used in which the coding sequences of an antigenicpeptide of the invention and any other desired sequences are flanked byregions that promote homologous recombination at a desired site in thegenome, thus providing for intrachromosomal expression of the nucleicacids encoding the antigenic peptide (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature342:435-438). In another particular embodiment, nucleic acid moleculesare used in which the coding sequences of an antigenic fusion protein ofthe invention and any other desired sequences are flanked by regionsthat promote homologous recombination at a desired site in the genome,thus providing for intrachromosomal expression of the nucleic acidsencoding the antigenic protein.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.); and WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).

In specific embodiments, viral vectors that contain nucleic acidsequences encoding antigenic peptides or antigenic fusion proteins areused. For example, a retroviral vector containing nucleic acid sequencesencoding an antigenic peptide or an antigenic fusion protein can be used(see, e.g., Miller et al., 1993, Meth. Enzymol. 217: 581-599). Theseretroviral vectors have been to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The nucleic acid sequences encoding antigenic peptides orantigenic fusion proteins to be used in gene therapy are cloned into oneor more vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6: 291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, human Gene Therapy 4: 129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3: 499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, human Gene Therapy5: 3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91: 225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2: 775-783. In apreferred embodiment, adenovirus vectors are used. Adeno-associatedvirus (AAV) has also been proposed for use in gene therapy (see, e.g,Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300; and U.S.Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a nucleic acidmolecule to cells in tissue culture by such methods as electroporation,lipofection, calcium phosphate mediated transfection, or viralinfection. Usually, the method of transfer includes the transfer of aselectable marker to the cells. The cells are then placed underselection to isolate those cells that have taken up and are expressingthe transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid molecule is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign nucleic acid molecules into cells (see, e.g., Loeffler and Behr,1993, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol.217: 618-644; Cline, 1985, Pharmac. Ther. 29: 69-92) and may be used inaccordance with the present invention, provided that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted. The technique should provide for the stable transfer of thenucleic acid to the cell, so that the nucleic acid is expressible by thecell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,subject's state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding the antigenic peptides or antigenicfusion proteins of the invention are introduced into the cells such thatthey are expressible by the cells or their progeny, and the recombinantcells are then administered in vivo for therapeutic effect. In aspecific embodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see e.g., PCT Publication WO 94/08598, dated Apr. 28, 1994;Stemple and Anderson, 1992, Cell 71: 973-985; Rheinwald, 1980, Meth.Cell Bio. 21A: 229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

The invention also relates to methods for producing an antigenic peptideof the invention or an antigenic fusion protein of the inventioncomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing anantigenic peptide or an antigenic fusion protein of the invention inwhich a host cell (i.e., a prokaryotic or eukaryotic cell) containing asuitable expression vector that includes a polynucleotide encoding anantigenic peptide or an antigenic fusion protein is cultured underconditions that allow expression of the encoded antigenic peptide or theencoded antigenic fusion protein. The antigenic peptide or the antigenicfusion protein can be recovered from the culture, conveniently from theculture medium, and further purified. The purified antigenic peptides orantigenic fusion proteins can be used in in vitro immunoassays which arewell known in the art to identify anti-IgE antibodies which bind to theantigenic peptides or the antigenic fusion proteins.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods arewell known in the art, as described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), incorporatedherein by reference. As used herein, an insect cell capable ofexpressing a polynucleotide of the present invention is “transformed.”

Alternatively, an antigenic peptide of the invention or an antigenicfusion protein of the invention may also be expressed in a form whichwill facilitate purification. For example, an antigenic peptide may beexpressed as fusion protein comprising a heterologous protein such asmaltose binding protein (MBP) glutathione-S-transferase (GST) orthioredoxin (TRX) which facilitates purification. Kits for expressionand purification of such fusion proteins are commercially available fromNew England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The protein can also be tagged with an epitopeand subsequently purified by using a specific antibody directed to suchepitope. One such epitope “Flag” is commercially available from Kodak(New Haven, Conn.).

The antigenic peptides of the invention or the antigenic fusion proteinsof the invention may also be expressed as a product of transgenicanimals, e.g., as a component of the milk of transgenic cows, goats,pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the antigenic peptide or theantigenic fusion protein.

Any method known to those of skill in the art can be used to produce anantigenic peptide or an antigenic fusion protein of the invention At thesimplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides. Theisolated antigenic peptides and antigenic fusion proteins of theinvention are useful, for example, in generating antibodies against thenative polypeptide.

One skilled in the art can readily follow known methods for isolatingpeptides and proteins in order to obtain one of the isolated antigenicpeptides or antigenic fusion proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, high performanceliquid chromatography (hPLC), reverse-phase high performance liquidchromatography (RP-hPLC), size-exclusion chromatography, ion-exchangechromatography, and immuno-affinity chromatography. See, e.g., Scopes,Protein Purification: Principles and Practice, Springer-Verlag (1994);Sambrook et al., in Molecular Cloning: A Laboratory Manual; Ausubel etal., Current Protocols in Molecular Biology.

An antigenic peptide or an antigenic fusion protein of the invention is“isolated” or “purified” when it is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus,protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of a contaminating protein. When an antigenic peptide or anantigenic fusion protein of the invention is recombinantly produced, itis also preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, 10%, or 5% of the volume of theprotein preparation. When an antigenic peptide or an antigenic fusionprotein of the invention is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the antigenic peptide or the antigenicfusion protein. Accordingly such preparations of the protein have lessthan about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the antigenic peptide or the antigenic fusionprotein.

The compositions of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays which can be used todetermine whether administration of a specific composition is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered acomposition, and the effect of such composition upon the tissue sampleis observed.

The expression of an antigenic peptide or an antigenic fusion proteincan be assayed by the immunoassays, gel electrophoresis followed byvisualization, or any other method known to those skilled in the art.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a composition has a desired effect upon such cell types. Inaccordance with the present invention, the functional activity of anantigenic peptide or an antigenic fusion protein can be measured by itsability to induce anti-IgE antibodies that inhibit IgE from binding toits receptor on mast cells or basophils in vitro without inducing therelease of vasoactive substances such as histamine.

Compositions for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to pigs,chicken, cows or monkeys.

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a composition ofthe invention to elicit the production of anti-IgE antibodies which donot cause anaphylaxis. In a preferred aspect, a composition of theinvention is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when thecomposition comprises a nucleic acid are described above; additionalappropriate formulations and routes of administration can be selectedfrom among those described herein below.

Various delivery systems are known and can be used to administer acomposition of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe composition, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262: 4429-4432), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intratumoral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, topical application, injection, or by meansof an implant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of an allergic reaction.

In another embodiment, a composition of the invention can be deliveredin a vesicle, in particular a liposome (see, e.g., Langer, 1990, Science249: 1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); and Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

In yet another embodiment, a composition of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see, e.g., Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; and Saudek et al.,1989, N. Engl. J. Med. 321: 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985,Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; and howardet al., 1989, J. Neurosurg. 71: 105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the composition of the invention is anucleic acid encoding an antigenic peptide or an antigenic fusionprotein of the invention, the nucleic acid can be administered in vivoto promote expression of its encoded antigenic peptide or antigenicfusion protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.USA 88: 1864-1868), etc. Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an antigenicpeptide or an antigenic fusion protein of the invention, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopela orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantigenic peptide or the antigenic fusion protein, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The antigenic peptides or antigenic fusion proteins of the invention canbe formulated as neutral or salt forms. Pharmaceutically acceptablesalts include those formed with free amino groups such as those derivedfrom hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment of cancer can be determined by known clinical techniques.In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will further depend on the route of administration and theseverity of the disease or disorder. however, suitable dosage ranges forintravenous administration are from about 20 to about 500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are from about 0.01 pg/kg body weight to about1 mg/kg body weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

EXAMPLES 1. Selection of Peptides and Conjugation to KLh

The CH3 domain of canine IgE as well as the junction between CH3 and CH4domains formed the basis for selection of peptide vaccine candidates.Various nested and overlapping peptides were selected using computerprograms for determination of appropriate antigenic properties includinghydrophilicity, surface probability, flexibility, antigenic index,amphiphilic helix, amphiphilic sheet, and secondary structures. Thepeptides were synthesized and conjugated to KLh at Zymed LaboratoriesInc. (San Francisco, Calif.) using cysteine-directed coupling method.The KLh-conjugated peptides were used to immunize rabbits at ZymedLaboratories Inc (South San Francisco). Peptides were also synthesizedat W. M. Keck Biotechnology Resource Center (New haven, Conn.) with anN-terminal biotin residue without conjugation to KLh to provide materialfor use in ELISA to detect anti-peptide antibodies induced in animalsimmunized with the KLh-peptide conjugates. Preferred peptides of thepresent invention include peptides of Seq. ID# 1 to Seq ID# 14 and theirhomologous sequences from other IgE species.

2: Reactivity of Rabbit Anti-peptide Antibodies with IgE-derivedPeptides

To test the ability of rabbit antisera to react with peptides of thepresent invention, an ELISA assay was developed as follows: BiotinylatedIgE peptides were diluted to five μg/ml in coating buffer (SodiumBicarbonate ph 9.0). Diluted peptides were added to the wells of aneutravidin plate (Pierce Chemical Co. Rockford, Ill.) at 100 μl/welland incubated at 4° C. overnight. Plates were washed 3× withphosphate-buffered saline containing 0.05% Tween-20 (PBST). Blockingbuffer (2% skim milk in PBST) was added to each well at 200 ul/well andthe plates were incubated at room temperature (RT) for 60 minutes.Plates were washed 3× with PBST. An 100 μl/well of 1:100 dilution ofappropriate rabbit antisera were added to the top row of the appropriatewells and serum samples diluted 10 fold to the appropriate plateposition. Plates were incubated at RT for 60 minutes. Plates were washed3× with PBST. An 100 μl/well of a 1:20,000 dilution of a horse-radishperoxidase conjugated goat anti-rabbit IgG (KPL Laboratories,Gaithersburg, Md.) were added to each well and the plates incubated atRT for 60 minutes. Plates were washed 3× with PBST. A 100 μl/well of TMBmicrowell substrate (KPL; Gaithersburg, Md.) was added to each well andthe plates incubated for 10-20 minutes at RT to allow for colordevelopment. The color development reaction was stopped with 50 μl/wellof 0.18M sulfuric acid. The optical density (O.D.) of all wells wasdetermined at wavelength of 450 nm using ELISA plate reader (Thermo Max;Molecular Devices, Sunnyvale, Calif.). As shown in FIG. 1, sera obtainedfrom rabbits after immunization with the indicated peptides had a muchhigher reactivity to the respective peptides than that obtained by serafrom rabbits prior to immunization with peptides of the presentinvention.

3. Reactivity of Rabbit Anti-peptide Antibodies with Canine IgE

Canine IgE monoclonal antibodies (Bethyl laboratories; Montgomery; Tex.)was dispensed in the wells of 96-well plates at 1 ug/well in a volume of100 μl. Plates were incubated at 4° C. overnight. Plates were washed 3×with PBST and 100 μl of blocking buffer (2% Skim milk in PBST) was addedto each well and incubated at room temperature (RT) for 60 minutes.Plates were washed 3× with PBST and 100 μl/well of 1:200 dilution ofappropriate rabbit antisera were added to the top row of the appropriatewells. Plates were incubated at RT for 60 minutes. Plates were washed 3×with PBST and 100 μl/well of a 1:10,000 dilution of a horseradishperoxidase conjugated goat anti-rabbit IgG (KPL Laboratories) was addedto all wells. Plates were incubated at RT for 60 minutes. Plates werewashed 3× with PBST and 100 μl/well of TMB substrate was added to eachwell. Color reaction was allowed to develop for 10-20 minutes. Colorreaction was stopped by adding 50 μl/well of 0.18M sulfuric acid.Optical density of all wells was determined at 450 nm in an ELISA platereader as above. As shown in FIG. 2, sera obtained from rabbits afterimmunization with the indicated peptides had a much higher reactivity tocanine IgE than that obtained by sera from rabbits prior to immunizationwith peptides of the present invention.

4. In Vitro Degranulation Inhibition Assay

The development of an IgE vaccine rests on the identification of IgEpeptides that induce antibodies which bind to soluble (free) IgE inserum and other bodily fluids, but do not cross-link receptor-bound IgEor release histamine from mast cells or basophils (i.e.,non-anaphylactogenic antibodies). In order to assess theanaphylactogenic potential of antibodies raised against nested oroverlapping sets of IgE-derived peptides such as those of the presentinvention, we developed an in vitro canine-specific degranulation assaybased on rat basophilic cell line RBL-2h3 transfected with the highaffinity receptor for canine IgE. When canine IgE is allowed to bind toits receptor on RBL2h3 cells and the receptor-bound IgE is incubatedwith anti-canine IgE antibodies, the receptor may be cross-linked (ifanti-IgE antibodies bind to receptor-bound IgE) and this receptorcross-linking results in the release of histamine from rat cells. Theamount of histamine released is a measure of the anaphylactic potentialof anti-IgE antibodies. Conversely, the lack of histamine releaseindicates that the anti-dog IgE antibodies do not cross-link receptorbound-IgE and the peptide that induced the formation of these antibodiesis suitable for use as a vaccine provided that the anti-peptideantibodies react with free IgE (e.g., IgE in serum or other fluids).Thus, the potential of any anti-IgE antibodies including antibodiesraised against peptides of the present invention to effect release ofhistamine would be easily measured using this assay.

The gene encoding the high affinity receptor for dog IgE was assembledby the Polymerase Chain Reaction at ATG laboratories Inc (Eden Prairie,Minn.) and cloned into the pCDNA6 expression vector (In Vitrogen; CA).Rat RBL2h3 cell line (ATCC, Rockville Md.) was transfected with thepCDNA6 plasmid containing the gene encoding the canine IgE receptorusing Fugene transfection reagent according to the manufacturer'srecommendation (Beohringer Mannheim). RBL-2h3 cell lines expressing thedog high affinity receptor was selected and maintained in mediacontaining 10 ug/ml blasticidin. The ability of canine IgE to bind tothe transfected rat cells was confirmed by various assays including Flowcytometry and cell-based ELISA.

5. Ascaris Sensitization and Immunization

The effect of vaccination with peptides of the present invention onIgE-mediated reactions was evaluated in a study of IgE-mediated skinwheal reactivity induced in animals following sensitization to ascarisextract. The study design is outlined in table 1 and the study wasconducted according to the following procedures:

1) Pre-sensitization procedures: Prior to commencement of the study(day-7), 5 ml of blood samples were collected from the jugular vein ofeach dog into serum separator tubes (SST). Serum was stored at −20 C.Skin tests were performed on all dogs by intradermal (ID) injection ofAsc-1 allergen (Greer laboratories). ID injections were carried out onthe shaved side/belly of each dog. Each animal received 6 injectionsrepresenting 10-fold serial dilution of Asc-1 allergen (50 μg-0.5 ng),one injection of 0.1 μg histamine (positive control) and one injectionof phosphate-buffered saline (PBS; negative control). Each injection isin a volume of 100 μl. Skin response was based on size of the area ofwheal reaction. The wheal area was outlined, and the maximal dimension(major axis) and the dimension perpendicular to that (minor dimension)in millimeter are multiplied to calculate the wheal area. Skin responseswere determined using metric rulers at 15 minutes following intradermalinjection of allergen. To help visualize the wheal reaction, each dogwas injected I/V with 5 ml of 1.0% solution of sterile Evan's blue dyeapproximately 5 minutes prior to skin tests.

2) Sensitization Schedule: Animals were injected with a mixture of 10 μgof Asc-1 and 2 mg of Rehydrogel (0.5 ml volume) and the mixture injectedsubcutaneously (S/C). At the same time, animals were injected with 500ng of Ricin (0.5 ml volume) intraperitoneally (I/P). The above injectionregimens was repeated 5 more times; once every 2 weeks. Asc-1 and Ricinwere dissolved in sterile PBS.

3) Post-sensitization skin test: Following the sensitization phase alldogs are evaluated for skin reactions. Skin tests were performed on alldogs as outlined under pre-sensitization skin test described above.

4) Vaccination: Animals in group F were not vaccinated. Animals ingroups A, B,C,D, and E were vaccinated as described in table X. Animalsare injected intramuscularly (I/M) with 1 ml of appropriate vaccinecontaining 50 μg of corresponding antigen.

5) Post-vaccination skin test: 14 days after the last vaccination, dogswere evaluated for skin reactions. Skin tests are performed on all dogsas outlined under pre-sensitization procedures.

6. Dog Anti-peptide Antibodies

The induction of antibodies in dogs vaccinated with specific peptides ofthis invention is evaluated with an ELISA assay as follows: Peptideswere diluted to 5 ug/ml in coating buffer (Sodium Bicarbonate ph 9.0)and dispensed at 100 μl/well of neutravidin plates (Pierce). Plates wereincubated at 4° C. overnight. Plates were washed 3× with PBST and 200 μlof blocking buffer (2% skim milk in PBST) was added to each well. Plateswere incubated at RT for 60 minutes. Plates were washed 3× with PBST and100 μl/well of 1:100 dilution of appropriate dog antisera was added tothe top row of the appropriate wells. Serum samples were then diluted 10fold to the appropriate plate position. Plates were incubated at RT for60 minutes. Plates were washed 3× with PBST and 100 μl/well of a1:20,000 dilution of a horse-radish peroxidase conjugated goat anti-dogIgG were added to each well. Plates were incubated at RT for 60 minutes.Plates were washed 3× with PBST and 100 μl/well of TMB substrate wasadded to all wells. Color reaction was allowed to develop for 10-20minutes at RT. Color reaction was stopped by adding 50 μl/well of 0.18Msulfuric acid and optical density was read at 450 nm in ELISA Reader asabove. As shown in table 2, sera obtained from dogs after immunizationwith the indicated peptides had a much higher reactivity to canine IgEthan that obtained by sera from dogs prior to immunization with peptidesof the present invention.

7. Skin Wheal Reactivity

The efficacy of peptides of the present invention in amelioratingIgE-mediated skin wheal reaction was determined by comparing the numberof vaccinated animals in which there was a reduction or completeremission of the skin wheal reaction relative to the skin wheal reactionof the same animals prior to vaccination. The skin wheal reactivity ofdogs following intradermal injection of ascaris extract is determined byinjection of 100 μl of serial 10 fold dilutions (50 μg to 0.5 ng) ofascaris extracts as well as PBS and histamine (0.1 μg/site). The size ofthe wheal reaction is determined as the product of the major and minoraxis of the wheal measured in millimeters using metric rulers. As can beseen from table 3, vaccination of animals with a cocktail of peptidesderived from the CH3/CH4 domains (SeqID1-4) result in complete remissionof skin wheal reaction in approximately 60% of animals.

TABLE 1 Experimental design Group Sensitization Vaccination # of dogs AAsc-1 RBS-1 (SEQ ID NO: 1) 7 B Asc-1 RBS-2 (SEQ ID NO: 2) 7 C Asc-1RBS-3 (SEQ ID NO: 3) 7 D Asc-1 RBS-4 (SEQ ID NO: 4) 7 E Asc-1 RBS-COC(SEQ ID NOS. 1-4) 7 F Asc-1 None (PBS) 7

TABLE 2 ELISA reactivity of dogs following immunization with IgEpeptides Group IgE peptide Pre-vaccination titer Post-vaccination titerA RSB-1 <100 1000 B RBS-2 <100 200 C RBS-3 <100 1000 D RBS-4 <100 1000 ERBS-COC <100 for RBS-1,2,3 1:1000 for RBS-1,2,3, and 4 and 4 F PBS <100100

TABLE 3 Skin wheal reactivity of dogs immunized with IgE peptides:Remission of Group Antigen skin wheal reaction A RBS-1 (SEQ ID NO: 1)0/7 B RBS-2 (SEQ ID NO: 2) 0/7 C RBS-3 (SEQ ID NO: 3) 2/7 D RBS-4 (SEQID NO: 4) 2/7 E RBS-COC (SEQ ID NOS.: 1-4 4/7 F None (PBS) 0/7

8. Food Allergy Model

In order to develop a food allergy model to evaluate the effect of theanti-IgE vaccines of the present invention, fifty dogs were sensitizedto ascaris antigens following injection of ascaris extract and ricin andthen challenged orally with ascaris extract as follows:

1. Pre-sensitization procedures: Skin tests were performed on all dogsby intradermal (ID) injection of Asc-1 allergen (Greer laboratories). IDinjections were carried out on the shaved side/belly of each dog. Eachanimal received 6 injections representing 10-fold serial dilution ofAsc-1 allergen (50 μg-0.5 ng), one injection of 0.1 μg histamine(positive control) and one injection of phosphate-buffered saline (PBS;negative control). Each injection is in a volume of 100 μl. Skinresponse was based on size of the area of wheal reaction. The wheal areawas outlined, and the maximal dimension (major axis) and the dimensionperpendicular to that (minor dimension) in millimeter are multiplied tocalculate the wheal area. Skin responses were determined using metricrulers at 15 minutes following intradermal injection of allergen. Tohelp visualize the wheal reaction, each dog was injected I/V with 5 mlof 1.0% solution of sterile Evan's blue dye approximately 5 minutesprior to skin tests

2. Sensitization Schedule: Animals were injected with a mixture of 10 μgof Asc-1 and 2 mg of Rehydrogel (0.5 ml volume) and the mixture injectedsubcutaneously (S/C). At the same time, animals were injected with 500ng of Ricin (0.5 ml volume) intraperitoneally (I/P). The above injectionregimens was repeated 4 more times, once every 2 weeks. Asc-1 and Ricinwere dissolved in sterile PBS.

3. Post-sensitization skin test: Following the sensitization phase alldogs were evaluated for skin reactions. Skin tests were performed on alldogs as outlined under pre-sensitization skin test described above.

4. Oral challenge: 14 days following the last skin test, dogs were given2 mg of ascaris extract dissolved in 1 ml of distilled water via theoral route. Dogs were observed for signs of food allergy includingvomiting and diarrhea. The results of oral challenge show thatapproximately 50% of sensitized dogs respond with clinical signs ofallergy with every oral challenge.

9. Flea Allergy Model

To evaluate the capacity of ricin to accelerate the development of fleaallergy dermatitis in dogs, a sensitization protocol in which dogs aresensitized to flea allergens in presence or absence of ricin isconducted as follows:

1. Five dogs are used as non-flea infested controls

2. Five dogs are exposed to fleas on a continual basis by infesting eachdog with 16 fleas on day 0 and then 16-17 more fleas every other day for12 weeks (last infestation day 84). Total flea exposure is 709 fleas.

3. Fifteen dogs are exposed to fleas episodically by infesting dogs with109 fleas on day 0 and then 100 fleas every other week for 12 weeks (709total fleas). Following a 48-hour infestation/exposure period fleas areremoved. This allows for a 12-day nonexposure period between eachreinfestation. Fleas are removed from dogs by the oral administration ofnitenpyram (Capstar: Novartis Animal health; dogs <11.4 kg administereda 11.4 mg tablet, dogs >11.4 kg administered a 57 mg tablet.) Studieshave determined that the product produces 100% mortality of fleas ondogs within 4 hours and is then rapidly eliminated from dogs with a T½(half-life) of 2.8 hours.

4. Fifteen dogs are exposed to fleas episodically by infesting dogs with109 fleas on day 0 and then 100 fleas every other week for 12 weeks (709total fleas). Following a 48-hour infestation/exposure period fleas areremoved. This allows for a 2-day nonexposure period between eachreinfestation. Fleas are removed from dogs by the oral administration ofnitenpyram as described above. In addition to flea exposure all dogsreceive an intraperitoneal injection of ricin (500 ng in 0.5 ml ofsterile saline from a 1 mg/ml stock solution) on day 0. Dogs not showinga significant rise in serum IgE titers (on Day 14) from presensitizationlevels (prior to flea exposure) are given a second injection of ricin onday 28. Ricin injection may be repeated as necessary to induceanti-Fleas IgE in dogs.

All of references cited herein are which incorporated herein in theirentirety by reference.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the claims.

1. An isolated antigenic peptide consisting of SEQ ID NO: 4 that inducesan anti-IgE immune response that does not cause anaphylaxis whenadministered to an animal.
 2. An isolated antigenic fusion proteincomprising a CH3 domain of an IgE molecule consisting of SEQ ID NO: 4that induces an anti-IgE immune response that does not cause anaphylaxiswhen administered to an animal; and a heterologous carrier protein fusedto said CH3 domain.
 3. A pharmaceutical composition for inducing ananti-IgE immune response that does not cause anaphylaxis comprising oneor more antigenic peptides of a CH3 domain of an IgE molecule whereinsaid one or more antigenic peptide consists of SEQ ID NO:
 4. 4. Apharmaceutical composition for inducing an anti-IgE immune response thatdoes not cause anaphylaxis comprising one or more antigertic peptides ofa CH3 domain of an IgE molecule wherein one or more of said antigenicpeptide consists of SEQ ID NO: 4; and a heterologous carrier proteinfused to one or more of said antigenic peptide.
 5. The pharmaceuticalcomposition of claim 4, wherein the heterologous carrier protein isselected from the group consisting of KLH, PhoE, rmLT, TraT, and gD fromBhV-1 virus.
 6. The pharmaceutical composition of claim 3 or 4, whereinthe anti-IgE immune response is the production of anti-IgE antibodieswhich bind to soluble IgE in serum and other bodily fluids, prevent IgEfrom binding to its high affinity receptors on mast cells and basophils,and do not cross-link receptor-bound IgE.
 7. The pharmaceuticalcomposition of claim 3 or 4 further comprising an adjuvant.
 8. Apharmaceutical kit comprising one or more containers filled with apharmaceutical composition of claim 3 or 4.